Arti Mahto

Genetic variation at the growth hormone (GH1) and growth hormone receptor (GHR) loci as a risk factor for hypertension and stroke

An increased prevalence of both hypertension and cerebrovascular stroke is apparent in growth hormone (GH) deficiency whilst hypertension is a frequent complication in acromegaly. This has suggested a possible link between GH, stature and arterial function. Since the risk of both hypertension and stroke also appears to be inversely correlated with adult height, we have instigated an exploratory study to assess whether inter-individual variation in the genes encoding human growth hormone (GH1) and the GH receptor (GHR) might be associated with an increased risk of hypertension and stroke. GH1 promoter haplotypes were found to differ significantly not only between hypertensive patients (n=111) and controls (n=121) but also between stroke patients (n=155) and controls (n=158). Intriguingly, the association between GH1 promoter haplotype and risk of hypertension was much greater in females than in males. An inverse correlation between height and central systolic blood pressure was apparent in both hypertensive patients and normal controls but was much stronger in individuals carrying at least one GH1 promoter risk haplotype. The GH1 genotype therefore constitutes a risk factor for hypertension that interacts with stature. A strong association was found between the presence of at least one GH1 risk haplotype and a family history of stroke at an early age (odds ratio: 9.07, 95% confidence interval: 1.14–72.22). Three novel GH variants (Arg16His, Phe176Cys, Cys189Arg) were identified during the course of this study. Although two exhibited markedly reduced biological activity in vitro, their clinical significance remains unclear. No association was found between GHR genotype and either hypertension or stroke, nor was any interaction noted between GHR and GH1 genotypes in terms of a disease association. However, an association between GHRd3 genotype and hypertension was observed among stroke patients, particularly females. Elevated HDL was found to be a risk factor for hypertension in individuals lacking a copy of the GHRd3 allele. Weak associations with GHR genotype were also noted for peripheral systolic and diastolic blood pressure in hypertensive patients. Although the underlying mechanisms are still unclear, our findings are consistent with a complex relationship between height, hypertension, GH1 promoter haplotype, GHR polymorphism and the risk of stroke.

Angiogenic gene expression and vascular density are reflected in ultrasonographic features of synovitis in early rheumatoid arthritis: an observational study

IntroductionNeovascularization contributes to the development of sustained synovial inflammation in the early stages of Rheumatoid Arthritis. Ultrasound (US) provides an indirect method of assessing synovial blood flow and has been shown to correlate with clinical disease activity in patients with Rheumatoid Arthritis. This study examines the relationship of US determined synovitis with synovial vascularity, angiogenic / lymphangiogenic factors and cellular mediators of inflammation in a cohort of patients with early Rheumatoid Arthritis (RA) patients prior to therapeutic intervention with disease modifying therapy or corticosteroids.MethodsAn ultrasound guided synovial biopsy of the supra-patella pouch was performed in 12 patients with early RA prior to treatment. Clinical, US and biochemical assessments were undertaken prior to the procedure. Ultrasound images and histological samples were obtained from the supra-patella pouch. Histological samples were stained for Factor VIII and a-SMA (a-smooth muscle actin). Using digital imaging analysis a vascular area score was recorded. QT-PCR (quantitative-PCR) of samples provided quantification of angiogenic and lymphangiogenic gene expression and immunohistochemistry stained tissue was scored for macrophage, T cell and B cell infiltration using an existing semi-quantitative score.ResultsPower Doppler showed a good correlation with histological vascular area (Spearman r - 0.73) and angiogenic factors such as vascular endothelial growth factor- A (VEGF-A), Angiopoietin 2 and Tie-2. In addition, lymphangiogenic factors such as VEGF-C and VEGF-R3 correlated well with US assessment of synovitis. A significant correlation was also found between power Doppler and synovial thickness, pro-inflammatory cytokines and sub-lining macrophage infiltrate. Within the supra-patella pouch there was no significant difference in US findings, gene expression or inflammatory cell infiltrate between any regions of synovium biopsied.ConclusionUltrasound assessment of synovial tissue faithfully reflects synovial vascularity. Both grey scale and power Doppler synovitis in early RA patients correlate with a pro-angiogenic and lymphangiogenic gene expression profile. In early RA both grey scale and power Doppler synovitis are associated with a pro-inflammatory cellular and cytokine profile providing considerable validity in its use as an objective assessment of synovial inflammation in clinical practice.

Evaluation of Minimally Invasive, Ultrasound-guided Synovial Biopsy Techniques by the OMERACT Filter — Determining Validation Requirements

Objective. Because limited data currently support the clinical utility of peripherally expressed biomarkers in guiding treatment decisions for patients with rheumatoid arthritis, the search has turned to the disease tissue. The strategic aim of the Outcome Measures in Rheumatology (OMERACT) synovitis working group over the years has been to develop novel diagnostic and prognostic synovial biomarkers. A critical step in this process is to refine and validate minimally invasive, technically simple, robust techniques to sample synovial tissue, for use both in clinical trials and routine clinical practice. The objective of the synovitis working group (SWG) at OMERACT 12 (2014) was to examine whether recently developed ultrasound (US)-guided synovial biopsy techniques could be validated according to the OMERACT filter for future clinical use recommendation. Methods The SWG examined whether current data reporting US-guided synovial biopsy of both large and small joints addressed the OMERACT filters of truth, discrimination, and feasibility. Results. There are currently limited data examining the performance of US-guided synovial biopsy, mainly from observational studies. Thus, it remains critical to evaluate its performance, within the clinical trials context, against the current gold standard of arthroscopic biopsy, with particular reference to: (1) synovial tissue yield, (2) capacity to determine treatment response as measured by a validated synovial biomarker, and (3) tolerability of the procedure. Conclusion. We summarize the discrete work packages agreed to as requirements to validate US-guided synovial biopsy and therefore lead to a global consensus on the use of synovial biopsy for research and clinical practice.

Mycophenolate mofetil inducing remission of lupus enteritis

We report the case of a young woman with a background history of discoid lupus who presented with abdominal pain, vomiting and intermittent diarrhoea. Physical examination revealed tenderness in the right upper quadrant with a palpable right inguinal lymph node without any other clinical signs of active lupus. Laboratory investigations showed normal inflammatory markers, positive ANA and Anti-Ro antibodies, persistent hypocomplementemia and lymphopenia, CT showed marked bowel oedema involving the small and large bowel (halo sign) with massive ascites and moderate right-sided pleural effusion. Mantoux test, AFB and TB cultures were negative. A diagnosis of lupus enteritis was made and treatment with high-dose steroids was commenced with little improvement. Treatment with cyclophosphamide was discussed but declined by the patient. Mycophenolate mofetil was commenced and resulted in significant clinical and radiological resolution. To the best of the authors’ knowledge this is the first report of the successful use of mycophenolate mofetil in inducing and maintaining remission in lupus enteritis.

OP0263 Synovial B-Cell Gene Signature Predicts Response To Rituximab Therapy

Background Predicting response to biologic therapies in RA remains a clinical challenge. The anti-CD20 monoclonal antibody Rituximab effectively depletes peripheral blood B-cells, however response rates are approximately 60%. We hypothesized that expression of CD20 in diseased tissue may be an important predictor of response, since up to 40% of patients have few/no B-cells present in the synovium. Objectives To ascertain whether a synovial B-cell gene signature can enhance prediction of responsiveness to Rituximab therapy and identify genes involved in response/non-response to treatment. Methods Synovial tissue was obtained using ultrasound-guided synovial biopsies from 20 patients with active RA who were treated with Rituximab therapy after failure of conventional DMARD and anti-TNF therapy. High-throughput quantitative real-time PCR for 190 genes was performed in collaboration with MedImmune (MedImmune, LLC) using the Fluidigm platform. Samples were classified as B-cell “rich” or “poor” according to levels of MS4A1 expression. Using an empirical Bayes statistical model, a further 17 significant (p<0.05) genes were identified to create a baseline B-cell gene expression signature. Hierarchical clustering and receiver operating characteristic curve analysis was used to assess ability to predict EULAR criteria response at 16 weeks. Gene expression was compared pre- and post-treatment in responders versus non-responders and correlated with delta DAS-28. Logistic regression with backward and forward selections using AIC was applied to identify further predictive models. Results Seventeen genes were identified that clustered with MS4A1 expression to create the B-cell signature. Fifteen of these are directly involved in B-cell and plasma-cell signaling or differentiation pathways and immunoglobulin synthesis. Two genes identified within this signature play a pivotal role in antigen presentation and TH17 differentiation. The baseline B-cell signature had an area under ROC curve (AUC) of 0.87 (95%CI 0.76–1.0) to predict response to Rituximab therapy at 16 weeks. Three additional genes were identified segregating responders from non-responders; that when combined with the baseline B-cell signature the AUC improved to 0.95. Additionally, ten genes were identified that significantly correlated with delta DAS-28. High expression of genes involved in the formation of ectopic lymphoid structures and the TH17 lineage correlated with non-response to Rituximab therapy (all p<0.05). Conclusions This study shows that a B-cell rich gene signature has high sensitivity to predict response to Rituximab therapy. High expression of TH17 related genes correlates with non-response to B-cell depletion, indicating that in a subset of patients, these pathways may play a more pivotal role in disease pathogenesis. The observed high expression of ELS associated genes in non-response may relate to variable synovial B-cell depletion; disruption of these follicles may be the key to response, particularly with repeat cycles. These findings warrant confirmation in a larger cohort of patients; however they strongly support the notion that a stratified approach to treatment relies upon the identification of biomarkers, both in the synovium and peripheral blood that reflect heterogenous molecular disease pathways in RA. Disclosure of Interest None declared

A7.6 Clinical and pathological differences of elderly- and younger-onset rheumatoid arthritis in an early arthritis cohort

Background and objectives Elderly-onset rheumatoid arthritis (EORA), starting after the age of 60, is a subset of disease with different characteristics from the classic younger-onset RA (YORA). We aimed to compare clinical and pathological features between these two disease subsets in the Barts Early Arthritis Cohort (BEAC). Material and methods We included BEAC patients fulfilling ACR 2010 criteria and assessed clinical and pathological variables at baseline and at 6 months of DMARD therapy. The primary outcome was the achievement of low disease activity (LDA) according to DAS28 at 6 months. We used multivariate logistic regression to determine predictors of LDA at 6 months. Results We included 140 patients, 99 YORA and 41 EORA. EORA patients were more frequently male (51.2 vs. 26.3, p = 0.004) and Caucasian (68.3 vs. 39.4, p = 0.02), had more proximal joint involvement at presentation (55.0 vs. 35.1, p = 0.031), more common abrupt (27.5 vs. 4.1, p < 0.001) and polymyalgia rheumatica (PMR)-like onset (23.7 vs. 0, p < 0.001), weight loss (15.0 vs. 4.1, p = 0.026), anaemia (48.6 vs. 28.4, p = 0.033), extra-articular manifestations (27.5 vs. 6.2, p = 0.004) and cardiovascular comorbidity (73.2 vs. 29.3, p < 0.001). At baseline, disease activity parameters, including ESR, CRP, joint counts, and composite scores (DAS28, CDAI and SDAI), were similar between both groups. Synovial pathotype classification (ectopic lymphoid-like structures positive (ELS+); diffuse myeloid infiltrate or non-inflammatory/pauci-immune) did not reveal differences between groups as a whole (p = 0.12). However, EORA patients presented pauci-immune pathotype less frequently (25.0 vs. 44.3, p = 0.045). Macrophage, B-cell, T-cell and plasmocyte semi-quantitative scores were also similar. At 6 months (n = 105), EORA (OR = 0.28, p = 0.047), female gender (OR = 0.23, p = 0.016), current smoking (OR = 0.11, p = 0.002) and high baseline DAS28 (OR = 0.41, p < 0.001) were negatively associated with reaching DAS28 LDA. Contrastingly, PMR-like onset was strongly predictive of DAS28 LDA (OR = 63.9, p = 0.004). Conclusions In an early arthritis cohort, EORA patients presented different clinical features at presentation, but similar synovial pathological characteristics. Adjusting for other significant factors such as gender, smoking, baseline DAS28 and PMR-like onset, EORA was negatively associated with the achievement of LDA at 6 months according to DAS28.

OP0032 Baseline Synovial B-Cell Status Predicts Response to Rituximab Therapy in RA

Background B-cells are key mediators in RA pathogenesis through the initiation of several pathways that lead to the perturbation of the immune system. Rituximab, an anti-CD20 monoclonal antibody is recommended for use after failure with traditional disease modifying agents and anti-TNF therapy. Despite using peripheral blood B-cells as a marker to ensure depletion, the clinical response to Rituximab remains variable, highlighting an unmet need for further biomarkers of response. Mechanisms that underpin the variation in clinical outcome are yet to be fully elucidated but may relate to distinct differences in cellular infiltrate and target expression within the synovium. Objectives The aim of this open-labelled pilot study was to test the hypothesis that patients with a B-cell rich versus B-cell poor synovial pathotype have an enhanced response to B-cell depletion following therapy with Rituximab. Methods Synovial biopsy (Ultrasound guided n=34, arthroscopic n=6) was performed at baseline in 40 patients with active RA (as defined by a DAS-28 score of >5.1) who had failed treatment with standard disease modifying therapy. 35 patients had received prior anti-TNF therapy, a subset of 5 patients were anti-TNF naïve. Rituximab was administered, with appropriate pre-medication, as two intravenous infusions at a dose of 1g 14 days apart. Synovial tissue was fixed in formalin, paraffin-embedded and cut to serial sections (3μm). Histological grading and lymphoid organization of the synovium was assessed by immunohistochemical analysis. After staining for CD20, the presence or absence of B-cells was determined by a semi-quantitative score (0-4)1. CD21 staining was carried out on samples with large aggregates to determine the presence of FDC in germinal centres (GC) within the synovium. Disease response to treatment (improvement in DAS-28>1.2) at 16 weeks was correlated with the presence or absence of B-cells and GC using Fishers exact test and Chi-squared testing, respectively. Results IHC for CD20 results were available from 39 patients. 29 [74%] patients were females, mean age 58.5 [29-80], 36 [92%] were anti-CCP positive and 32 [82%] were RF positive. 12 [30%] showed a response (DAS-28 >1.2) improvement at 3 months. 24 [62%] patients had little or no B-cell infiltrate within the synovium. In this subgroup, 20 [83%] did not exhibit a significant clinical response. (p=0.031). In B-cell rich non-responders, the presence of GC was noted in 5 out of 6 individuals [83%]; only 1 patient who was GC positive responded to Rituximab. (p=0.01, n=37) Conclusions This open-labeled pilot study strongly suggests that response to Rituximab therapy is determined by the presence or absence of B-cells within the synovium. In patients with B-cell rich synovium, the presence of GC appears to be an important marker of resistance to Rituximab therapy. This pilot study also highlights the potential to utilize synovial pathotype as a biomarker to predict disease outcomes and response. References Kraan, M.C., Haringman, J.J., Post W.J., Versendaal, J., Breedveld, F.C., Tak. P.P. Immunohistological analysis of synovial tissue for differential diagnosis in early arthritis. Rheumatology 1999; 38:1074–1080. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5609

AB0132 Presence of Synovial Lymphocyte Aggregates Correlates with A More Severe Clinical Phenotype in Patients with Early Inflammatory Arthritis

Background Although many observations have suggested that ectopic lymphoid structures within the inflamed synovium are immunologically functional and can support chronic inflammation, their clinical significance is still controversial. Moreover, most of data comes from patients with long standing disease activity, while data in early arthritis is still scarce. In particular, it is unclear whether the presence of lymphocytic aggregates within the synovium correlates with clinical phenotype and degree of inflammation in early inflammatory arthritis patients. Objectives The aim of this study is to evaluate the correlation between synovial lymphocytic aggregates with clinical phenotype in an early inflammatory arthritis cohort. Methods 155 disease-modifying antirheumatic drug-naïve patients with early inflammatory arthritis (<12 months duration, at least 1 swollen joints) has been recruited at Barts and The London Hospital. Baseline disease characteristics assessment included erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, rheumatoid factor (RF), anti-cyclic-citrullinated peptide (CCP) and 28 joints-disease activity score (DAS28). Ultrasound-guided synovial biopsy has been obtained for all patients. Sections of paraffin embedded synovial tissue were stained with standard Haematoxylin and Eosin (H&E) and graded as either a diffuse or aggregate synovitis. The degree of lymphoid organisation was further assessed by immnohistochemical staining of sequentially cut sections. The number of lymphocytic aggregates was counted in each section and graded according to a validated, previously published grading system [Manzo, A. et al; European Journal of Immunology2005]. Results The presence of lymphocytic aggregates has been found in almost 30% of patients; Lymphocytic aggregates are associated with increased inflammatory markers and DAS28, as well as RF and CCP seropositivity (p<0.05); The number of B cells and plasma cells in the synovium of early rheumatoid arthritis patients is closely associated (p<0.05), suggesting in situ differentiation. Conclusions The presence of lymphocytic aggregates within the synovium in early arthritis correlates with a more severe clinical phenotype, including inflammatory markers, disease activity and antibody status. Further studies are needed in order to establish the clinical relevance of this association, and in particular its potential prognostic value. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5836